Respiratory distress syndrome (RDS), also known as hyaline membrane disease, is a major cause of morbidity and mortality of the prematurely born infant. RDS is believed to be caused primarily by a deficiency of lung surfactant--a lipid-protein mixture which coats the airspaces of the lung--thereby reducing surface tension and preventing airspace collapse. The principal component of lung surfactant--dipalmitoylphosphatidylcholine (DPPC)--was identified several years ago (Klaus et al., Proc Natl Acad Sci USA (1961) 47:1858; Avery et al., Am J Dis Child (1959) 97:517). It is believed that administration of lung surfactant to an individual having or at risk of developing RDS is a desirable therapy, and the literature discloses various clinical studies of therapeutic administration of different lung surfactant preparations.
Mammalian lung surfactant is a complex material containing primarily phospholipids and associated proteins or apolipoproteins. The literature contains various lung surfactant protein preparations, including those with DPPC. Generally, these preparations include natural human surfactant (purified from human amniotic fluid--Merrit et al. N Eng J Med (1986) 315:787); semisynthetic surfactant (prepared by combining DPPC and high-density lipoprotein--Halliday et al. Lancet (1984) 1:476); animal lung surfactant (isolated by organic extraction of the whole lung or lavage fluid--Kwong et al. Pediatrics (1985) 76:585); and purified human surfactant apoproteins (SP-A, SP-B, and/or SP-C purified from natural sources or derived by recombinant DNA technology (Jobe et al. Am Rev Resp Dis (1987) 136:1032, Glasser et al. J Biol Chem (1988) 263:10326, PCT Publication WO 88/05820, and PCT Publication WO 90/01540, and which may be reconstituted with surfactant lipids (Revak et al. J Clin Invest (1987) 81:826).
Significant progress has been made in the purification and characterization of the human surfactant apoproteins. However, different investigators have observed variation in the number of peptides with apparent molecular masses between 5 and 18 kDa, and their nominal molecular masses. Besides the molecular weight variations, researchers have experienced difficulty in separating the proteins isolated from native sources from their lipid components. Conventional lipid extraction procedures generally fail to separate the proteins completely from the surfactant lipids. One procedure which addresses this problem is described by Arjomaa and Hallman (Ann Biochem (1988) 171:207-212). That process involves a two-step purification of a human chloroform/methanol extract containing the SP4-6 peptide using a Sep Pak Florisil column, followed by reversed-phase high-pressure liquid chromatography (HPLC).
Recombinant production of surfactant proteins obviously does not require separation of protein from surfactant lipids. However, because microbially produced surfactant proteins can be expressed at a fairly high level, the high level of expression causes the recombinant protein to precipitate intracellularly in the form of refractile bodies, which do require further isolation and purification. The problem addressed by the present invention is how to safely and efficiently recover recombinant surfactant protein from the cell in a purified, renatured form that is acceptable for clinical use.
Solvent systems used both in the extraction of lung surfactant proteins and in subsequent chromatographic procedures typically involve combinations of non-polar solvents and buffers such as ether/ethanol, ether/chloroform, and chloroform/methanol. Many of these non-polar agents are quite toxic; for example, chloroform has been shown to be carcinogenic in rats. The use of such solvents in the purification of compounds and compositions intended for pharmaceutical applications therefore raises product safety concerns about potential toxicity in humans. It is, therefore, an object of the present invention to provide a process for isolating and purifying recombinant lung surfactant protein which avoids the use of chloroform-containing solvents and buffers. The use of substantially non-toxic solvents permits one to employ extraction and chromatographic procedures heretofore not used in the purification of surfactant proteins.